Antibody Binding Assay for Malaria Antigens

Antibody binding to PcCSP or PcTRAP in macaque plasma were measured by an IgG enzyme linked immunosorbent assay (ELISA) using previously described methods [18 (link),19 (link),20 (link)]. Briefly, Nunc Maxisorp Immuno ELISA plates coated with PcCSP or PcTRAP antigens diluted in PBS to a final concentration of 2 µg/mL and incubated at room temperature (RT) overnight. Plates were washed 6 times with PBS/0.05% Tween (PBS/T) and blocked with 300 µL with PierceTM protein-free (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA, U.S.) for 2 h at RT. Macaque plasma was added and serially diluted 3-fold down in PBS/T with 50 µL per well as final volume and incubated for 2 h at RT. Following washing 6 times with PBS/T, bound antibodies were detected following a 1 h incubation with 50 µL of alkaline phosphatase-conjugated antibodies specific for monkey IgG (Sigma Aldrich, SLM, U.S.). Following additional 6 washes with PBS/T, development was achieved using 100 µL of 4-nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at OD405 were measured and c using a CLARIOstar instrument (BMG Labtech, Aylesbury, GB). Log reciprocal antibody titers were defined by an absorbance value three standard deviations greater than the average OD405 of the control. The antibody titers were analyzed by unpaired t-test to determine the p-values.